ESPE Abstracts

Background: Loss of imprinting has been implicated in the pathogenesis of several human diseases. Monogenic causes of central precocious puberty (CPP) were identified in families with loss-of-function mutations in two paternally expressed imprinted genes: Makorin zinc finger 3 (MKRN3) and Delta-like 1 homolog (DLK1). The role of imprinting defects in CPP has not been described so far.

Objective: To investigate the methylation status at primary differentially methylated regions (DMR) of MKRN3 and DLK1 in a cohort of CPP patients.

Patients and Methods: Fifty-six CPP girls (48 sporadic, 8 familial) were selected for analysis. They had normal hypothalamic-pituitary region MRI. Leukocyte DNA was obtained from all patients. MKRN3 and DLK1 pathogenic allelic variants were initially excluded by DNA sequencing analysis. Bisulfite treatment followed by Allele-Specific Methylated Multiplex Real-Time Quantitative PCR was performed with leukocyte DNA, analyzing separately the methylation index (MI) of MKRN3:TSS-DMR and DLK1/MEG3:IG-DMR for each patient. Results were compared with MI of 50 adult controls, 15 prepubertal controls girls and 18 pubertal controls girls with normal pubertal development.

Results: Mean age at puberty onset was 6.1±1.9yr for all CPP girls. Hypomethylation at DLK1/MEG3:IG-DMR was identified in two patients with sporadic CPP (patients I and II), confirming a molecular diagnosis of Temple syndrome. Interestingly, both girls had been firstly referred to pediatric endocrinology for presenting precocious menarche. During follow-up, other clinical findings were noticed: being born small for gestational age, prominent forehead, small hands/feet, overweight and hyperinsulinemia or early type 2 diabetes. SNP array was performed in patient I, identifying a partial uniparental disomy at chromosome 14 (upd(14)). On the other hand, patient II had normal CGH array and microsatellites analysis, excluding copy number variations and upd(14), and indicating a mechanism of epimutation. In the remaining patients, mean MI for DLK1/MEG3:IG-DMR was 49±1.5%. In all patients, mean MI for MKRN3:TSS-DMR was 49±5%. Besides that, as a group, there were no significant correlations between age at puberty onset and: 1) MI for MKRN3:TSS-DMR (P=0,69) and 2) MI for DLK1/MEG3:IG-DMR (P=0,45).

Conclusion: There were no leukocyte DNA methylation defects at MKRN3 imprinting control region. DLK1/MEG3:IG-DMR hypomethylation was identified in two patients with CPP. Loss of effective imprinting of DLK1 locus may be a mechanism involved in the etiology of CPP, and should be investigated in patients presenting additional findings of Temple syndrome.